Potential of animal-welfare compliant and sustainably sourced serum from pig slaughter blood.

The animal product most used as a stimulatory additive for cell cultivation is still fetal bovine serum (FBS). Besides the ethical concerns regarding serum collection, the main problems of FBS are batch-to-batch variability and the resulting risk of lower reproducibility, the differences between species, the presence of undefined/unknown components, and the risk of contamination. In contrast, pig blood, which is a by-product of slaughter, is a sufficiently available and sustainable resource with a high degree of standardization in terms of donor age, weight, and genetics. The variations in preparations from pig slaughter blood seem to be comparatively low, and consequently, batch effects might be much smaller, suggesting that the reproducibility of the research data obtained may be increased. Our pilot study aimed to investigate, as a proof of concept, whether adult human and porcine stem cells of different tissue origins proliferate and differentiate adequately when FBS is completely or partially replaced by porcine serum (PS). We could show that the human and porcine stem cells were vital and proliferated under partial and full PS supplementation. Furthermore, using PS, the two cell types studied showed tissue-specific differentiation (i.e., lipid vacuoles as a sign of adipogenic or myotubes as a sign of myogenic differentiation). In conclusion, the pig slaughter blood-derived serum has promising potential to be a replacement for FBS in adult stem cell cultures. Therefore, it could serve as a basis for the development of new cell culture supplements.

Use of a type 1 genetic risk score for classification of diabetes type in young Australian adults: the Fremantle Diabetes Study Phase II.

The applicability of a UK-validated genetic risk score (GRS) was assessed in 158 participants in the Fremantle Diabetes Study Phase II diagnosed between 20 and <40 years of age with type 1 or type 2 diabetes or latent autoimmune diabetes of adults (LADA). For type 1 versus type 2/LADA, the area under the receiver operating characteristic curve (AUC) was highest for serum C-peptide (0.93) and lowest for the GRS (0.66). Adding age at diagnosis and body mass index to C-peptide increased the AUC minimally (0.96). The GRS appears of modest diabetes diagnostic value in young Australians.

Serum vitamin B12, distal symmetrical polyneuropathy and anaemia in type 2 diabetes: the Fremantle Diabetes Study Phase 2.

BACKGROUND: There are limited data relating to the effects of metformin-associated vitamin B12 deficiency on the risk of distal symmetrical polyneuropathy (DSPN) and megaloblastic anaemia in well-characterised community-based cohorts. AIMS: To assess inter-relationships between metformin therapy, vitamin B12 deficiency assessed using serum active B12 concentrations, and DSPN and anaemia in 1492 Fremantle Diabetes Study Phase 2 (FDS2) participants with type 2 diabetes. METHODS: Prevalence rates of vitamin B12 deficiency (total <80 pmol/L, active <23 pmol/L) and borderline deficiency (total ≥80 and ≤200 pmol/L, active ≥23 and ≤35 pmol/L) were determined using baseline sera. The relationship between vitamin B12 status and both DSPN and anaemia was assessed using multivariable analyses. RESULTS: Most FDS2 participants (94.4%) were vitamin B12 replete (total serum concentration >200 pmol/L, active >35 pmol/L), 2.0% were deficient (total <80 pmol/L, active <23 pmol/L) and the remainder (3.6%) borderline. Although metformin treatment increased the odds of deficiency (4.2%, 3.1% borderline) in a dose-dependent fashion (odds ratio (95% confidence interval) 39.4 (4.90-316) for >2000 mg daily compared with no treatment; P < 0.001), there was no significant association between vitamin B12 status and DSPN, anaemia (haemoglobin ≤130 g/L males, ≤120 g/L females), haemoglobin concentration or mean corpuscular volume (P ≥ 0.147). Metformin increased the likelihood of anaemia, especially at high doses, independent of vitamin B12 deficiency. CONCLUSIONS: Since nutritional sources likely attenuate metformin-associated vitamin B12 malabsorption and its clinical sequelae in developed countries such as Australia, there is no need for routine/opportunistic serum vitamin B12 screening in metformin-treated patients.

Cold Atmospheric Pressure Plasma-Activated Medium Modulates Cellular Functions of Human Mesenchymal Stem/Stromal Cells In Vitro.

Cold atmospheric pressure plasma (CAP) offers a variety of therapeutic possibilities and induces the formation of reactive chemical species associated with oxidative stress. Mesenchymal stem/stromal cells (MSCs) play a central role in tissue regeneration, partly because of their antioxidant properties and ability to migrate into regenerating areas. During the therapeutic application, MSCs are directly exposed to the reactive species of CAP. Therefore, the investigation of CAP-induced effects on MSCs is essential. In this study, we quantified the amount of ROS due to the CAP activation of the culture medium. In addition, cell number, metabolic activity, stress signals, and migration were analyzed after the treatment of MSCs with a CAP-activated medium. CAP-activated media induced a significant increase in ROS but did not cause cytotoxic effects on MSCs when the treatment was singular and short-term (one day). This single treatment led to increased cell migration, an essential process in wound healing. In parallel, there was an increase in various cell stress proteins, indicating an adaptation to oxidative stress. Repeated treatments with the CAP-activated medium impaired the viability of the MSCs. The results shown here provide information on the influence of treatment frequency and intensity, which could be necessary for the therapeutic application of CAP.

Assessment of venous pressure by compression sonography of the internal jugular vein during 3 days of bed rest.

Compression sonography has been proposed as a method for non-invasive measurement of venous pressures during spaceflight, but initial reports of venous pressure measured by compression ultrasound conflict with prior reports of invasively measured central venous pressure (CVP). The aim of this study is to determine the agreement of compression sonography of the internal jugular vein (IJVP) with invasive measures of CVP over a range of pressures relevant to microgravity exposure. Ten healthy volunteers (18-55 years, five female) completed two 3-day sessions of supine bed rest to simulate microgravity. IJVP and CVP were measured in the seated position, and in the supine position throughout 3 days of bed rest. The range of CVP recorded was in line with previous reports of CVP during changes in posture on Earth and in microgravity. The correlation between IJVP and CVP was poor when measured during spontaneous breathing (r = 0.29; R2  = 0.09; P = 0.0002; standard error of the estimate (SEE) = 3.0 mmHg) or end-expiration CVP (CVPEE ; r = 0.19; R2  = 0.04; P = 0.121; SEE = 3.0 mmHg). There was a modest correlation between the change in CVP and the change in IJVP for both spontaneous ΔCVP (r = 0.49; R2  = 0.24; P < 0.0001) and ΔCVPEE (r = 0.58; R2  = 0.34; P < 0.0001). Bland-Altman analysis of IJVP revealed a large positive bias compared to spontaneous breathing CVP (3.6 mmHg; SD = 4.0; CV = 85%; P < 0.0001) and CVPEE (3.6 mmHg; SD = 4.2; CV = 84%; P < 0.0001). Assessment of absolute IJVP via compression sonography correlated poorly with direct measurements of CVP by invasive catheterization over a range of venous pressures that are physiologically relevant to spaceflight. However, compression sonography showed modest utility for tracking changes in venous pressure over time. NEW FINDINGS: What is the central question of this study? Compression sonography has been proposed as a novel method for non-invasive measurement of venous pressures during spaceflight. However, the accuracy has not yet been confirmed in the range of CVP experienced by astronauts during spaceflight. What is the main finding and its importance? Our data show that compression sonography of the internal jugular vein correlates poorly with direct measurement of central venous pressures in a range that is physiologically relevant to spaceflight. However, compression sonography showed modest utility for tracking changes in venous pressure over time.

Novel integrated workflow allows production and in-depth quality assessment of multifactorial reprogrammed skeletal muscle cells from human stem cells.

Skeletal muscle tissue engineering aims at generating biological substitutes that restore, maintain or improve normal muscle function; however, the quality of cells produced by current protocols remains insufficient. Here, we developed a multifactor-based protocol that combines adenovector (AdV)-mediated MYOD expression, small molecule inhibitor and growth factor treatment, and electrical pulse stimulation (EPS) to efficiently reprogram different types of human-derived multipotent stem cells into physiologically functional skeletal muscle cells (SMCs). The protocol was complemented through a novel in silico workflow that allows for in-depth estimation and potentially optimization of the quality of generated muscle tissue, based on the transcriptomes of transdifferentiated cells. We additionally patch-clamped phenotypic SMCs to associate their bioelectrical characteristics with their transcriptome reprogramming. Overall, we set up a comprehensive and dynamic approach at the nexus of viral vector-based technology, bioinformatics, and electrophysiology that facilitates production of high-quality skeletal muscle cells and can guide iterative cycles to improve myo-differentiation protocols.

Oxidative Stress Response in Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells.

Reactive oxygen species (ROS) can irreversibly damage biological molecules, a process known as oxidative stress. Elevated ROS levels are associated with immune cell activation. Sustained immune system activation can affect many different cells in the environment. One cell type that has been detected in almost all tissues of the body is mesenchymal stem/stromal cells (MSC). MSC possess proliferation and differentiation potential, thus facilitating regeneration processes. However, the regenerative capacity of MSC might be impaired by oxidative stress, and the effects of long-term oxidative stress on MSC functions are sparsely described. The examination of oxidative stress is often performed by exposure to H2O2. Since H2O2 is rapidly degraded, we additionally exposed the cell cultures to glucose oxidase (GOx), resulting in sustained exposure to H2O2. Using these model systems, we have focused on the effects of short- and long-term oxidative stress on viability, migration, differentiation, and signaling. All cellular functions examined were affected by the applied oxidative stress. The differences that occur between pulsed and sustained oxidative stress indicated higher oxidative stress in MSC upon direct H2O2 exposure, whereas the GOx-induced prolonged exposure to H2O2 seems to allow for better cellular adaptation. The mechanisms underlying these different responses are currently unknown.

A robust multiplex immunoaffinity mass spectrometry assay (PromarkerD) for clinical prediction of diabetic kidney disease.

BACKGROUND: PromarkerD is a novel proteomics derived blood test for predicting diabetic kidney disease (DKD). The test is based on an algorithm that combines the measurement of three plasma protein biomarkers (CD5L, APOA4, and IBP3) with three clinical variables (age, HDL-cholesterol, and eGFR). The initial format of the assay used immunodepletion of plasma samples followed by targeted mass spectrometry (MRM-LCMS). The aim of this study was to convert the existing assay into an immunoaffinity approach compatible with higher throughput and robust clinical application. METHODS: A newly optimised immunoaffinity-based assay was developed in a 96 well format with MRM measurements made using a low-flow LCMS method. The stability, reproducibility and precision of the assay was evaluated. A direct comparison between the immunoaffinity method and the original immunodepletion method was conducted on a 100-person cohort. Subsequently, an inter-lab study was performed of the optimised immunoaffinity method in two independent laboratories. RESULTS: Processing of plasma samples was greatly simplified by switching to an immunoaffinity bead capture method, coupled to a faster and more robust microflow LCMS system. Processing time was reduced from seven to two days and the chromatography reduced from 90 to 8 min. Biomarker stability by temperature and time difference treatments passed acceptance criteria. Intra/Inter-day test reproducibility and precision were within 11% CV for all biomarkers. PromarkerD test results from the new immunoaffinity method demonstrated excellent correlation (R = 0.96) to the original immunodepletion method. The immunoaffinity assay was successfully transferred to a second laboratory (R = 0.98) demonstrating the robustness of the methodology and ease of method transfer. CONCLUSIONS: An immunoaffinity capture targeted mass spectrometry assay was developed and optimised. It showed statistically comparable results to those obtained from the original immunodepletion method and was also able to provide comparable results when deployed to an independent laboratory. Taking a research grade assay and optimising to a clinical grade workflow provides insights into the future of multiplex biomarker measurement with an immunoaffinity mass spectrometry foundation. In the current format the PromarkerD immunoaffinity assay has the potential to make a significant impact on prediction of diabetic kidney disease with consequent benefit to patients.

Prevalence and incidence of thyroid dysfunction in type 1 diabetes, type 2 diabetes and latent autoimmune diabetes of adults: The Fremantle Diabetes Study Phase II.

OBJECTIVE: Since the results of published studies assessing thyroid dysfunction complicating diabetes have been variable in quality, inconsistent and may not reflect contemporary clinical care, the aim of this study was to determine its prevalence and incidence in a large, well-characterized, representative cohort. DESIGN: Community-based, longitudinal, observational study. PATIENTS: A total of 1617 participants from the Fremantle Diabetes Study Phase II (FDS2), including 130 (8.0%) with type 1 diabetes, 1408 (87.1%) with type 2 diabetes, and 79 (4.9%) with latent autoimmune diabetes of adults (LADA). MEASUREMENTS: Serum thyrotropin (TSH) and free thyroxine (FT4) at baseline between 2008 and 2011 and in those attending Year 4 follow-up. RESULTS: The prevalence of known thyroid disease (ascertained from baseline self-reported thyroid medication use or hospitalization data) was 11.7% (189/1617). Of the remaining 1428 participants, 5.1% (73/1428) had biochemical evidence of subclinical hypothyroidism, 1.1% (15/1428) overt hypothyroidism, 0.1% (2/1428) subclinical hyperthyroidism and 0.2% (3/1428) overt hyperthyroidism, representing an overall baseline prevalence of thyroid disease of 17.4% (282/1617). During 5694 patient-years of follow-up, 25 (3.0%) of the 844 with a normal baseline TSH and follow-up data developed known thyroid disease. Of the remaining 819, 3.4% developed subclinical hypothyroidism, 0.2% overt hypothyroidism and 0.5% subclinical hyperthyroidism. There were no statistically significant differences in the prevalence or incidence of thyroid dysfunction by diabetes type. CONCLUSIONS: Thyroid dysfunction, known or detected through screening, is common in diabetes. These data suggest the need for periodic clinical and biochemical screening for thyroid disease in all types of diabetes.

Human adipocytes and CD34+ cells from the stromal vascular fraction of the same adipose tissue differ in their energy metabolic enzyme configuration.

Adipose tissue plays a role in energy storage and metabolic balance and is composed of different cell types. The metabolic activity of the tissue itself has been a matter of research for a long time, but comparative data about the energy metabolism of different cell types of human subcutaneous adipose tissue are sparse. Therefore, we compared the activity of major energy metabolic pathways of adipocytes and CD34+ cells from the stromal vascular fraction (SVF) separated from the same tissue. This CD34+ cell fraction is enriched with adipose tissue-derived mesenchymal progenitors, as they account for the largest proportion of CD34+ cells of the SVF. Adipocytes displayed significantly higher mitochondrial enzyme capacities compared to CD34+ SVF-cells, as shown by the higher activities of isocitrate dehydrogenase and ß-hydroxyacyl-CoA dehydrogenase. Inversely, the CD34+ SVF-cells showed higher capacities for cytosolic carbohydrate metabolism, represented by the activity of glycolysis and the pentose phosphate pathway. Thus, the CD34+ SVF-cells may ensure the provision of pentose phosphates and reduction equivalents for the replication of DNA during proliferation. The data indicate that these two cell fractions of the human adipose tissue vary in their metabolic configuration adapted to their physiological demands regarding proliferation and differentiation in vivo.

Protein Network Analysis Identifies Changes in the Level of Proteins Involved in Platelet Degranulation, Proteolysis and Cholesterol Metabolism Pathways in AECOPD Patients.

Chronic obstructive pulmonary disease (COPD) is characterised by a progressive pulmonary and systemic inflammation. Acute exacerbations of COPD (AECOPD) are associated with acute inflammation and infection, increase in the rates of morbidity and mortality. Previous proteomic studies have focussed on identifying proteins involved in COPD pathogenesis in samples collected from the lung (e.g. lung tissue biopsies, bronchoalveolar lavage and sputum) but not from blood of patients who experienced AECOPD. In this study, plasma was analysed by two independent quantitative proteomics techniques; isobaric tag for relative and absolute quantitation (iTRAQ) and multiple reaction monitoring (MRM) to identify differential expression of circulating proteins in patients with stable COPD (sCOPD) and AECOPD. Firstly, iTRAQ performed on pooled plasma samples from AECOPD, sCOPD, and healthy non-smoking controls (HC) revealed 15 differentially expressed proteins between the 3 groups. MRM subsequently performed on a separate cohort of AECOPD, sCOPD, and HC patients confirmed 9 proteins to be differentially expressed by AECOPD compared to HC (Afamin, alpha-1-antichymotrypsin, Apolipoprotein E, Beta-2-glycoprotein 1, Complement component C9, Fibronectin, Immunoglobulin lambda like polypeptide 5, Inter-alpha-trypsin inhibitor heavy chain H3, Leucine rich alpha-2-glycoprotein 1). Network analysis demonstrates that most of these proteins are involved in proteolysis regulation, platelet degranulation and cholesterol metabolism. In conclusion, several potential plasma biomarkers for AECOPD were identified in this study. Further validation studies of these proteins may elucidate their roles in the development of AECOPD.

Energy metabolic capacities of human adipose-derived mesenchymal stromal cells in vitro and their adaptations in osteogenic and adipogenic differentiation.

Mesenchymal stromal/stem cells (MSC) are important in tissue homeostasis and regeneration due to their ability for self-renewal and multipotent differentiation. Differentiation, as well as proliferation, requires adaptations in the cell metabolism. However, only few data exist concerning the energy metabolism of non-differentiating and differentiating MSC. In this study we compared capacities of major energy metabolic pathways of MSC from human adipose tissue (adMSC) in vitro in the non-differentiated state with those of osteogenically or adipogenically differentiating adMSC. To this end we quantified the proliferation and differentiation status of adMSC and analyzed maximum enzyme capacities and several enzyme isoforms of major energy metabolic pathways regarding their activity and gene expression. We could show that non-differentiating and osteogenic cultivation conditions induced proliferation and showed increasing capacities of the glycolytic marker enzyme phosphofructokinase as well as the marker enzyme of the pentose phosphate pathway glucose-6-phosphate dehydrogenase. Adipogenic stimulation, which was accompanied by the absence of proliferation, reduced the glycolytic capacity (e.g. decreased glyceraldehyde 3-phosphate dehydrogenase capacity) and induced an increase in mitochondrial enzyme capacities. These changes in energy metabolism might represent an adaptation of adMSC to the high energy demand during proliferation and to the specific cellular functions during osteogenic or adipogenic differentiation respectively.

Analysis by proteomics reveals unique circulatory proteins in idiopathic pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease that has a poor 3-year median survival rate with unclear pathophysiology. Radiological features include bibasal, subpleural fibrosis and honeycombing while its pathology is characterized by fibroblastic foci and honeycombing. Proteomic analysis of circulating molecules in plasma may identify factors that characterize IPF and may assist in the diagnosis, prognostication and determination of pathogenic pathways in this condition. METHODS: Two independent quantitative proteomic techniques were used, isobaric tags for relative and absolute quantitation (iTRAQ) and multiple reaction monitoring (MRM), to identify differentially expressed plasma proteins in a group of IPF patients in comparison to healthy controls with normal lung function matched for age and gender. RESULTS: Five proteins were identified to be differentially expressed in IPF compared to healthy controls (upregulation of platelet basic protein and downregulation of actin, cytoplasmic 2, antithrombin-III, extracellular matrix protein-1 and fibronectin). CONCLUSION: This study further validates the combinational use of non-targeted discovery proteomics (iTRAQ) with targeted quantitation by mass spectrometry (MRM) of soluble biomarkers to identify potentially important molecules and pathways for pulmonary diseases such as IPF.

Lipopolysaccharide induces proliferation and osteogenic differentiation of adipose-derived mesenchymal stromal cells in vitro via TLR4 activation.

Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation of adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.

Prevalence of diabetes in Australia: insights from the Fremantle Diabetes Study Phase II.

BACKGROUND: Accurate diabetes prevalence estimates are important for health service planning and prioritisation. Available data have limitations, suggesting that the contemporary burden of diabetes in Australia is best assessed from multiple sources. AIMS: To use systematic active detection of diabetes cases in a postcode-defined urban area through the Fremantle Diabetes Study Phase II (FDS2) to complement other epidemiological and survey data in estimating the national prevalence of diabetes and its types. METHODS: People with known diabetes in a population of 157 000 were identified (n = 4639) from a variety of sources and those providing informed consent (n = 1668 or 36%) were recruited to the FDS2 between 2008 and 2011. All FDS2 participants were assigned a type of diabetes based on clinical and laboratory (including serological and genetic) features. Data from people identified through the FDS2 were used to complement Australian Health Survey and National Diabetes Services Scheme prevalence estimates (the proportions of people well controlled on no pharmacotherapy and registering with the National Diabetes Services Scheme respectively) in combination with Australian Bureau of Statistics data to generate the prevalence of diabetes in Australia. RESULTS: Based on data from multiple sources, 4.8% or 1.1 million Australians had diabetes in 2011-2012, of whom 85.8% had type 2 diabetes, 7.9% type 1 diabetes and 6.3% other types (latent autoimmune diabetes of adults, monogenic diabetes and secondary diabetes). CONCLUSIONS: Approximately 1 in 20 Australians has diabetes. Although most have type 2 diabetes, one in seven has other types that may require more specialised diagnosis and/or management.

The prevalence of monogenic diabetes in Australia: the Fremantle Diabetes Study Phase II.

OBJECTIVE: To determine the prevalence of monogenic diabetes in an Australian community. DESIGN: Longitudinal observational study of a cohort recruited between 2008 and 2011. SETTING: Urban population of 157 000 people (Fremantle, Western Australia). PARTICIPANTS: 1668 (of 4639 people with diabetes) who consented to participation (36.0% participation). MAIN OUTCOME MEASURES: Prevalence of maturity-onset diabetes of the young (MODY) and permanent neonatal diabetes in patients under 35 years of age, from European and non-European ethnic backgrounds, who were at risk of MODY according to United Kingdom risk prediction models, and who were then genotyped for relevant mutations. RESULTS: Twelve of 148 young participants with European ethnic backgrounds (8%) were identified by the risk prediction model as likely to have MODY; four had a glucokinase gene mutation. Thirteen of 45 with non-European ethnic backgrounds (28%) were identified as likely to have MODY, but none had a relevant mutation (DNA unavailable for one patient). Two patients with European ethnic backgrounds (one likely to have MODY) had neonatal diabetes. The estimated MODY prevalence among participants with diagnosed diabetes was 0.24% (95% confidence interval [CI], 0.08-0.66%), an overall population prevalence of 89 cases per million; the prevalence of permanent neonatal diabetes was 0.12% (95% CI, 0.02-0.48%) and the population prevalence 45 cases per million. CONCLUSIONS: One in 280 Australians diagnosed with diabetes have a monogenic form; most are of European ethnicity. Diagnosing MODY and neonatal diabetes is important because their management (including family screening) and prognosis can differ significantly from those for types 1 and 2 diabetes.

Analysis of migration rate and chemotaxis of human adipose-derived mesenchymal stem cells in response to LPS and LTA in vitro.

Mesenchymal stem cells (MSC) are able to stimulate the regeneration of injured tissue. Since bacterial infections are common complications in wound healing, bacterial pathogens and their components come into direct contact with MSC. The interaction with bacterial structures influences the proliferation, differentiation and migratory activity of the MSC, which might be of relevance during regeneration. Studies on MSC migration in response to bacterial components have shown different results depending on the cell type. Here, we analyzed the migration rate and chemotaxis of human adipose-derived MSC (adMSC) in response to the basic cell-wall components lipopolysaccharide (LPS) of Gram-negative bacteria and lipoteichoic acid (LTA) of Gram-positive bacteria in vitro. To this end, we used transwell and scratch assays, as well as a specific chemotaxis assay combined with live-cell imaging. We found no significant influence of LPS or LTA on the migration rate of adMSC in transwell or scratch assays. Furthermore, in the µ-slide chemotaxis assay, the stimulation with LPS did not exert any chemotactic effect on adMSC.

Binding of indocyanine green in polycaprolactone fibers using blend electrospinning for in vivo laser-assisted vascular anastomosis.

BACKGROUND AND OBJECTIVE: The clinical application of laser-assisted vascular anastomosis is afflicted by unreliable and low bonding strengths as well as tedious handling during microvascular surgery. The challenge to be met arises from the flow-off of the chromophore during soldering that changes the absorption and stains the surrounding tissue, leading to an uncontrollable thermal damage zone. In this study, we investigated the feasibility to produce an indocyanine green (ICG)-loaded patch by electrospinning and tested its applicability to both in vitro and in vivo microvascular laser soldering. MATERIALS AND METHODS: A blend of polycaprolactone and ICG was electrospun to produce a pliable patch. Prior to soldering, the patch was soaked in 40% wt. bovine serum albumin solution. The solder patch was wrapped in vitro around blood vessel stumps of rabbit aortas. An intraluminal balloon catheter enabled an easy alignment and held the setup in place. The soldering energy was delivered via a diffusor fiber from the vessel lumen using a diode laser at 810 nm. During the procedure, the surface temperature was observed with an infrared camera. Afterward, samples were embedded in methylmethacrylate and epon to study thermal damage. The quality of the fusion was assessed by measuring the tensile strength. After in vitro tests with rabbit aortas, eight large white pigs were subjected to an acute in vivo experiment, and the artery of the latissimus dorsi flap was anastomosed to the distal femoral artery. RESULTS: The ICG-loaded patch, produced by electrospinning, has a thickness of 279 ± 62 µm, a fiber diameter of 1.20 ± 0.19 µm, and an attenuation coefficient of 1,119 ± 183 cm-1 at a wavelength of 790 nm. The patch was pliable and easy to handle during surgery. No leakage of the chromophore was observed. Thermal damage was restricted to the Tunica adventitia and Tunica media and the area of the vessel wall that was covered with the patch. Six pigs were successfully treated, without any bleeding and with a continuous blood flow. The in vivo flap model yielded a similar tensile strength compared to in vitro laser-assisted vascular anastomoses (138 ± 52 vs. 117 ± 30 mN/mm2 ). CONCLUSION: Our study demonstrated the applicability of the ICG-loaded patch for laser-assisted vascular anastomosis. By using electrospinning, ICG could be bound to polymer fibers, avoiding its flow-off and the staining of the surrounding tissue. This patch demonstrated several advantages over liquid solder as it was easier to apply, ensured a high and reliable bonding strength while maintaining a constant concentration of ICG concentration during the surgery. Lasers Surg. Med. 49:928-939, 2017. © 2017 Wiley Periodicals, Inc.

Prevalence and prognosis of a low serum testosterone in men with type 2 diabetes: the Fremantle Diabetes Study Phase II.

BACKGROUND: Because published studies have usually involved imprecise assays and selected patients with limited additional data and follow-up, the consequences of a low serum testosterone in diabetes are unclear. This study assessed the prevalence, associates and prognosis of a low testosterone in community-dwelling men with type 2 diabetes. DESIGN: Longitudinal observational study. PATIENTS: 788 men (mean ± SD age: 65·8 ± 11·3 years) followed for 4·0 ± 1·1 years. MEASUREMENTS: Serum testosterone, SHBG, erectile dysfunction (ED; Sexual Health Inventory for Men score <22), anaemia (haemoglobin <130 g/l), all-cause mortality. RESULTS: The mean ± SD total serum testosterone by liquid chromatography/mass spectrometry was 13·1 ± 5·9 nmol/l (30·6% <10 nmol/l). Most men with a total testosterone <10 nmol/l (67·0%) had a normal/low serum LH. Serum testosterone was independently associated with anaemia (P < 0·001), but not ED (P = 0·80), in logistic regression models. The optimal cut-point (Youden Index) for anaemia was 9·8 nmol/l (sensitivity 53·6%, specificity 75·4%). During the follow-up, 102 men (12·9%) died. There was a U-shaped relationship between total serum testosterone quintiles and death (P = 0·003, log rank test). The middle quintile (>11·1 to ≤13·7 nmol/l) had the lowest risk and there was a 78% increased risk for highest (>16·9 nmol/l) vs lowest (≤8·6 nmol/l) quintile in Cox proportional hazards modelling (P = 0·036). Free serum testosterone and SHBG quintiles were not associated with death. CONCLUSIONS: These data provide some support for the general conventional serum testosterone <10 nmol/l cut-point in identifying an increased risk of anaemia and the subsequent death in men with type 2 diabetes, but indicate that high-normal levels are also an adverse prognostic indicator.

Serum bicarbonate concentration and the risk of cardiovascular disease and death in type 2 diabetes: the Fremantle Diabetes Study.

BACKGROUND: Serum bicarbonate is associated with mortality, heart failure (HF) and progression of renal failure in studies of healthy people and patients with chronic kidney disease, but the significance of these observations in unselected patients with diabetes in the general population is unknown. The aim of this study was to determine whether serum bicarbonate was associated with mortality and cardiovascular disease risk in type 2 diabetes. METHODS: Baseline serum bicarbonate was available for 1283 well-characterized community-based patients (mean ± SD age 64.1 ± 11.3 years, 48.7 % males) from the longitudinal observational Fremantle Diabetes Study followed for a mean of 12 years. Associations between serum bicarbonate and mortality, coronary heart disease (CHD) and incident HF were analysed using Cox proportional hazards regression. RESULTS: Serum bicarbonate was independently and negatively associated with incident CHD. For each 1 mmol/L increase in bicarbonate, the hazard ratio for CHD was 0.95 (95 % confidence interval 0.92-0.99) after adjustment for age as time scale, age at baseline, sex, English fluency, diabetes duration, loge(serum triglycerides), loge(urinary albumin: creatinine ratio), peripheral sensory neuropathy and peripheral arterial disease. There were no independent associations between serum bicarbonate and all-cause mortality [0.98 (0.95-1.004)] or incident HF [0.99 (0.95-1.03)]. CONCLUSIONS: Serum bicarbonate was a significant independent predictor of incident CHD but not death or HF in community-based patients with type 2 diabetes. This supports intervention trials of bicarbonate replacement in type 2 patients at risk of CHD and who have a low serum bicarbonate concentration.